Mitochondria pelleted together with nuclei from 293 Cells using Sucrose gradient

I am doing an assay that requires the isolation of transfected plasmid DNA in the nuclei from 293(HEK) cells. The goal was to exclude everything from the cytoplasm, except the plasmid in the nuclei. So,what I did was to lyse and isolate the nuclei following this modified sucrose gradient protocol(below) and then isolate the plasmid DNA from the nuclei using miniprep (Fermentas), treating them as if they're bacterial cells. It's a alkaline lysis kit for bacterial plasmid. Is this method workable?

According to the protocol, only nuclei should be able to make it through the sucrose cushion layer and form a pellet. But from running the miniprep isolation on a gel, it seems that I am getting huge DNA band at about 12-16kbp length, which was suspected to be mitochondria. Could this indicate that the sucrose gradient separation was carried out improperly?

I would really appreciate your help very much!

Protocol:

1) Cells on a 10cm plate was washed twice with 5ml cold PBS, scrapped and transfered into 15 ml tube.2) Centrifuge about 800g for about 2 minutes to pellet the cells.3) Remove PBS and resuspend in 4ml Sucrose Buffer I.4) Incubate on ice for 20 minutes5) Add 4ml Sucrose Buffer II into the nuclei.6) Layer the ultracentrifuge tube with 2ml Sucrose Buffer I, then the nuclei sample, then topped off with 1ml Sucrose Buffer I7) Ultracentrifuge for 30,000g for 45 minutes8) Vacuum aspirate the supernatant and resuspend in 100ul glycerol storage buffer.9) Carry out plasmid isolation using Fermentas miniprep.

I don't like this method. you are incubating in Buffer I with 1% Triton for 20 min. That is too long. I would use digitonin instead of Triton or NP40. Look for Afshin Samali's method for rapid isolation of cytoplasm with digitonin on google. That method gave me pure cell fractions.

My lab doesn't have NP40 and I read that it was okay to replace the NP40 with double strength Triton. http://www.protocol-...posts/5421.html So I was hoping it would work. I will try lessen the incubation time then?

I took a quick look into the method and it seems like the mitochondrial is pelleted together as well in that method right? I may not have described it clearly in the title but I only want the nuclei fraction, excluding the mitochondrial fraction. I apologize if I misunderstood the protocol. Thanks again!

No, what I'm saying is that NP40 and Triton X-100 are both too strong and they might break down any cell component. You need to study all these detergents very well. There are plenty of textbooks describing each one of them. For fractionation usually people use milder detergents. I found digitonin a very reliable detergent. Last time I even tried Tween 20 but I couldn't fractionate well. However, it depends on the protocol too. I would guarantee Afshin Samali's method. It really works, and it fractionates fast.